It is the long term general objective of this proposal and of my laboratory to understand gene regulation through poly(A) site use in complex transcription units. For all pol II transcription units, 3' end formation and transcription termination are mandatory steps in mRNA biosynthesis and in the definition of the transcription unit. As we are defining gene regulation by 3' end processing, we are necessarily also considering how 3' end processing affects i.) pol II transcription termination ii.) splice site recognition and utilization and iii.) transport of mRNA from the nucleus to the cytoplasm. Each of these steps is recognized as playing an increasingly large role in the strategy of eucaryotic gene regulation. The specific goal of this research plan is to extend our characterization of the molecular biology and biochemistry of 3' end formation and transcription termination in RNA pol II transcription units. We are characterizing regulation of poly(A) site use in tandem poly(A) site containing transcription units to understand how poly(A) site choice is determined for complex transcription units. These studies necessarily require a comparison to function of individual poly(A) sites. The efficiency of 3' processing for a given substrate pre-mRNA is a major focus of these studies since it is a key determinant in alternative processing as well as in transcription termination. We have shown that recognition and presumably cleavage efficiency at the poly(A) site is a required first step in formation of a "termination competent" RNA polymerase II elongation complex. We are extending our characterization of 3' processing to a characterization of how 3' processing causes formation of the termination competent elongation complex. We are pursuing two parallel and complementing strategies for characterization of 3' processing and transcription termination; i. in vivo studies which we are using to identify functional parameters which are operating to regulate 3' processing and termination within the cell ii. in vitro studies defining the biochemistry of 3'processing and transcription termination.